Method for continuous Immunoassay monitoring

ABSTRACT

The present invention relates to assays that employ an enzyme label or tag that acts on a substrate by obtaining electrons from an electrode (electrocatalysis) and an apparatus for use in such assays. In particular the present invention provides an assay and apparatus for multiple or continuous monitoring of the amount of analyte in a sample or sample source without requiring regeneration of the measuring electrode or its associated reagents.

[0001] The present invention relates to assays that employ an enzymelabel or tag that acts on a substrate by obtaining electrons from anelectrode (electrocatalysis) and an apparatus for use in such assays.

BACKGROUND OF THE INVENTION

[0002] Immunoassay techniques are based on the ability of antibodies toform complexes with the corresponding antigens or haptens. This propertyof highly specific molecular recognition of antigens by antibodies leadsto high selectivity of assays based on immune principles. The highaffinity of antigen-antibody interactions results in great sensitivityof immunoassay methods. The use of a label or indicator to verify thatan antigen/antibody interaction has occurred is the basis forimmunoassay methods.

[0003] Immunoassay techniques have been used mainly in clinical analysesand medical diagnostics. However, immunoassay applications in otherareas such as environmental control, food quality control, etc. areexpanding. Certain limitations in assaying techniques due to existingprocedures have limited somewhat the expansion into such other areas.

[0004] In this respect, during the last few years a significant numberof publications have dealt with non-conventional (alternative)immunoassay techniques designed to expand the accuracy or applicabilityof immunoassays. In most cases the development of alternativeimmunoassay techniques aims at improvements in performance ofconventional immunoanalysis. Often such improvement attempts aredirected to decreasing analysis times, increasing assay sensitivity, andsimplifying and automating assay procedures.

[0005] For example, the utilization of enzymes able to catalyzeelectrochemical reactions by direct (mediatorless) mechanism(bioelectrocatalysis) would allow for the detection ofimmuno-interactions in real time. Such applications ofbioelectrocatalysis in the development of immunosensors are based on theself-assembling or displacement of molecule/label complexes or“molecular transducers” on the surface of an electrode that has beenmodified by immunospecies that bind the complex. Ordinarily theseimmunospecies would be complimentary to the immunoconjugate whichincludes the electrocatalytically active enzyme-label.

[0006] Antigen immobilized on the electrode surface interacts with theenzyme-labeled antibody which results in the attachment of the enzyme tothe electrode surface. Attachment of the electrocatalytic active enzymeon the electrode surface initiates, in the presence of a substrate, anelectrocatalytic reaction. Therefore, the formation of anantigen-labeled antibody complex on the electrode surface is accompaniedby an assembling of the molecular transducing layer. The rate ofelectron transfer can be limited by the efficiency of electricalconnection between the enzyme-label and the electrode surface, which isalready modified by the immobilized immunospecies.

[0007] A potentiometric immunosensor based on mediatorlessbioelectrocatalysis has been utilized which employed laccase enzyme asan electrocatalyst-label. The electrocatalytic property of the enzyme inthe reaction of oxygen electroreduction (reaction 1) allowed thedetection of the biospecific interaction of a laccase-labeled receptor,or antibody, with a ligand modified electrode. Formation of a complexbetween the laccase labeled antibody and antigen on the electrodesurface results in a considerable shift in electrode potential due tothe catalytic reduction of over voltage. Analysis was performed in acompetitive scheme, and a single measurement was made with 20 minutes.Such a potentiometric immunoassay does not require an electrochemicallyactive mediator. The reaction substrates were atmospheric oxygen andelectrons that were transferred directly from the electrode to theoxygen molecule via the active site of the enzyme. Insulin was used as amodel analyte.

[0008] In the above immunoassay sensor, the electron which is the“second substrate” of enzymatic reaction can be captured by theenzyme-label only from the electrode surface. Therefore, only moleculesintimately attached to the electrode surface generate electrochemicalsignal. The rate of attachment of electrocatalyst molecules to theelectrode surface is proportional to the rate of formation of theimmuno-complex on the electrode surface. The rate of attachment ofelectrocatalyst molecules to the electrode surface is proportional tothe rate of formation of the immuno-complex on the electrode surface.The rate of immunointeraction on the electrode surface can be directlymonitored by amperometric or potentiometric mode.

[0009] However, assays based on mediatorless bioelectrocatalysis arelimited in that primarily one of the two assay procedures set forthbelow are utilized and only a single assay measurement may be takenbefore the electrode is regenerated or replaced by a new electrode. Thetwo competitive assay procedures are:

[0010] (a) Competitive Immunoassay With an Initial Label-freeElectrode—An electrode having no attached analyte/enzyme label isutilized as a starting point and a measured amount of analyte mediaalong with a measured quantity of analyte/enzyme label are assayed by acompetitive binding assay procedure. After maximum association with theelectrode has occurred the amperometric or potentiometric measure resultis compared to that of an electrode having 100% analyte/enzyme labelassociated. The difference in measurements corresponds proportionally tothe amount of analyte in the media being assayed. and

[0011] (b) Displacement Immunoassay With an Initial Label-loadedElectrode—An electrode having the maximum amount of attachedanalyte/enzyme label (a fully loaded electrode) is utilized as astarting point and a measured amount of analyte media is assayed by acompetitive binding assay procedure. After maximum displacement of theanalyte/enzyme label from the electrode by the analyte of the media hasoccurred the amperometric or potentiometric measure result is comparedto that of the initial fully loaded electrode (having 100%analyte/enzyme label associated). The difference in measurementscorresponds proportionally to the amount of analyte in the media beingassayed.

[0012] In addition to the laccase enzyme label, the potentiometricimmunosensor employing peroxidase as an electrocatalyst-label has alsobeen developed. The basic principle is the same as for the laccase basedimmunosensor. The electrode surface is modified by an immobilizedantigen (rabbit IgG). The peroxidase-antibody conjugate associates withthe antigen on the electrode surface. Once added to the media, and onreaching the electrode surface, the antibody-conjugated peroxidasestarts to catalyze the electro-reduction of hydrogen peroxide. Thisresults in an increase (anodic shift) in the electrode potential.

[0013] Both the laccase and peroxidase label immunosensors based onbio-electrocatalytic detection (as discussed above) allow directdetection of immunointeraction in real time. However, these sensors mustbe regenerated or replaced (e.g., disposable sensors) after eachmeasurement. Accordingly, such immunoassay procedures do not allowcontinuous monitoring of the analyte. In addition, such procedures are amulti-stage process that result in a general complexity of analysis andrequire a highly qualified technician to conduct the assay.

[0014] Accordingly, there is a need for immunoassay procedures that canbe continuous, particularly automatable procedures or procedures that donot require highly qualified technicians to conduct the assay.

SUMMARY OF THE INVENTION

[0015] An object of the present invention is to provide an improvedbioelectrocatalysis immunoassay apparatus for detecting an analyte, theapparatus comprising a sensing device with an electrode, wherein thesensing device has the ability to monitor changes in the amount ofanalyte in a liquid without requiring regeneration or replacement of theelectrode or of the reagents used in the assay. Preferably, the sensingdevice of the apparatus is capable of multiple intermittent and/orcontinuous immunoassay measurements of the same analyte without arequirement for regeneration or replacement of the electrode or otherreagents.

[0016] In one aspect the apparatus is for detecting an analyte that isan antigen, antibody or hapten by use of a labeled detection compoundthat is labelled with an electrocatalytic enzyme, wherein the labeleddetection compound is either the analyte or the binder for the analyte,and wherein the apparatus provides an electrode to which is permanentlyaffixed (i) a binder for the analyte in the case wherein the labeleddetection compound is a labeled analyte or (ii) the analyte in the casewherein the labeled detection compound is a labeled binder for theanalyte. If such detection compound becomes closely associated with theelectrode of the apparatus, the electrocatalytic enzyme label or tagwill interact with the electrode to cause a detectable electrical changefor the electrode. In one embodiment the analyte is the same antigen,antibody or hapten as the detection compound (wherein the detectioncompound is labeled with an electrocatalytic enzyme or tag) and some ofthe fixed amount of the labelled detection compound complex that isdetectable by the electrode due to bioelectrocatalysis competes with theanalyte for binding to the binder of the electrode. Alternatively, thebinder on the electrode and the analyte are the same antigen, antibodyor hapten and compete for binding to the detection compound that islabelled with the electrocatalytic enzyme.

[0017] In accordance with the above apparatus (depending upon whetherthe analyte is the same as the binder or the same as the detectioncompound) the analyte will bind to either the binder or to the detectioncompound. In either event, the amount of the detection compound thatattaches to the binder is inversely proportional to the concentration ofthe analyte in the sample being analyzed. The amount of the detectioncompound which is bound to the binder of the electrode is indirectlymeasurable by bioelectrocatalysis and the amount of analyte which ispresent in the sample is therefore detectable by the amount ofbioelectrocatalysis occurring at the electrode's surface. In each of theabove cases (i.e., (i) when the binder and analyte are the same and (2)when the binder and the detection compound are the same, the electrodeonly transfers an electron to the label as a substrate when the labeleddetection compound is attached to the binder affixed to the electrode.Therefore, a shift in potential or current, can be observed by theamount of labeled detection compound that binds to the binder on theelectrode, which amount of labeled detection compound bound to thebinder on the electrode is inversely proportional to the amount ofanalyte in the sample. When current is measured instead of potential atthe electrode, the current is proportional to the amount of laccaselabel attached to the electrode. Measurement of potential is preferredsince amperometric measurement of the electrode signal takes intoconsideration the surface area of the electrode and the density of thelaccase label attached to the electrode. Thus, potential measures areusually more accurate, but sometimes amperometric measures are moreaccurate and should be used. The ordinary practicioner in this fieldwould know when to use a particular electrode type. Thus, when the terms“potentiometric electrode” and “potentiometric assay” and the like areused in this application and claims the word “ampherometric” may besubstituted for “potentiometric”.

[0018] A further object of the invention is to provide an improvedbioelectrocatalysis immunoassay sensing device wherein the electrode isencased by a housing member comprising at least one porous orsemi-porous surface, such as a semipermeable membrane, that is permeablefor an analyte and impermeable to the labelled detection compound,preferably a detection compound that is labelled with anelectrocatalytic enzyme. In a preferred aspect the sensing devicecomprises a particular quantity of the labelled detection compound whichis enclosed within the housing member, which labelled detection compoundmay contact the electrode when the sensing device is placed in a liquidor gaseous medium and the at least one porous or semi-porous surface isimpermeable to the labelled detection compound.

[0019] A preferred object of the invention is to provide an apparatusfor immunodetermination of target analyte (antigen, antibody or hapten)in an analyte sample, where said apparatus comprises

[0020] (a) a sensing device comprising:

[0021] (i) a potentiometric working electrode at least one surface ofwhich is located within said housing member, wherein said electrode isconnected to a potentiometric measuring circuit and said electrode hasthe ability to provide an electron to an enzyme label which will deliverthe electron to a first substrate for the enzyme label, and saidelectrode has permanently affixed to at least one of its surfaces abinder for at least a detection compound which is labelled with anelectrocatalytic enzyme;

[0022] (ii) a housing member comprising at least one surface that ispermeable to an analyte and impermeable to the labelled detectioncompound, wherein the detection compound is a member selected from thegroup consisting of (a) a binder for the analyte (in the case where thebinder on the electrode is the analyte) and (b) the analyte (in the casewhere the binder on the electrode is a binder for the analyte), and ineach case the detection compound is labelled with an electrolytic enzymeor tag; and

[0023] (iii) an internal media which is located within the housingmember and which is a gel or liquid containing a pre-determined amountof labelled detection compound as herein above described, or capable ofcontaining a predetermined amount of the labelled detection compound;and

[0024] (b) an electrochemical reference electrode connected to apotentiometric measuring circuit,

[0025] and wherein the internal media of (iii) or the analyte samplecomprises a second substrate for the enzyme label or tag, such asoxygen. The analyte will either bind to the binder or to the detectioncompound.

[0026] In a preferred aspect, the apparatus further comprises at leastone measuring device which is connected directly or indirectly to thesensing device and/or the reference electrode. Preferably, the at leastone measuring device is a member selected from a digital voltmeter orother similar measuring device. In one aspect said measuring device isinterfaced with a personal computer as well as the sensing element andthe electrochemical reference probe. In a preferred aspect, themeasuring device is also connected to a member selected from (i) asignal recorder which is an X-T recorder, (ii) a microprocessor baseddata acquisition system with a digital display, and (iii) a personalcomputer.

[0027] Additionally, an object of this invention is provide a sensingdevice comprising an external housing with at least one semipermeablesurface and having within the housing device at least one surface of aworking electrode comprised of an electrode body made byelectrochemically inert electro-conductive material modified by a binderimmobilized on its surface which will bind to at least a detectioncompound which is labelled with an electrocatalytic enzyme, and thebinder may be the same as the analyte or may be a binder for theanalyte. In a preferred aspect, the binder is a binder for both theanalyte and the labeled detection compound, whereby the binder willreversibly bind individually to the analyte or to the labelled detectioncompound which will each compete to be bound by the binder. Preferably,the detection compound is labelled with the laccase enzyme which can useoxygen and electrons from the electrode as substrates.

[0028] Another object of the invention is a sensing element whichcomprises (a) a potentiometric working electrode, (b) an externalhousing with at least one surface that is a semipermeable surface (suchas a diffusion membrane) through which an analyte may diffuse and isimpermeable to at least one detection compound which is labelled with anelectrolytic enzyme or tag and the detection compound will bind toeither the binder of the electrode, or to both the binder on theelectrode and the analyte of the sample, and (c) an electrochemicalreference electrode. In a preferred aspect the sensing element furthercomprises an internal media, which is a gel or liquid, and a fixedquantity of the labelled detection compound, or comprises a means forinserting a quantity of labelled detection compound and/or the internalmedia comprising a fixed quantity of the labelled detection compound.

[0029] Another object of the invention is to provide a portablebiosensor capable of continuously detecting a target analyte in a widerange which operates in a continual potentiometric mode from whichmultiple potentiometric measurements are available that correspond tothe amount of analyte present in a given sample being assayed.

[0030] In yet another aspect, an object of the present invention is toprovide a method for intermittently or continuously conductingimmunoassay measurements wherein a plurality of different measurementsfor an analyte are available for a single electrode without requiringregeneration of said electrode and other reagents. In particular, suchobject is accomplished by a method of determination a target analytebased on displacement activity of the target analyte and apotentiometric mode comprising the following steps:

[0031] (a) immersing of the intermediate and/or continuousbioelectrocatalysis immunoassay sensing element (described above) in aassay medium containing the target analyte,

[0032] (b) allowing the target analyte of the assay medium to diffusethrough the diffusion membrane of the sensing device and travel to thesurface of the working electrode,

[0033] (c) permitting an immuno-equilibrium to be established within thesensing device with respect to the amount of target analyte present inthe assay medium due to displacement of some or all of a labelleddetection compound from the binder on the surface of the electrode bythe target analyte which target analyte becomes bound to the binder onthe surface of the sensing element in the case where the detectioncompound is the same as the analyte or the target analyte becomes boundto the detection compound in the case where the detection compound is abinder for the analyte,

[0034] (d) measuring at least one shift of the electrode potentialcaused by the displacement of some or all of the labeled detectioncompound from the electrode's surface and the resulting diminishment orabsence of electrocatalytic properties caused by the label of detectioncompound,

[0035] (e) determining the potentiometric sensor response which isproportional to the degree of displacement of the labeled detectioncompound from the binder on the surface of the working electrode causedby competitive binding of the target analyte, and

[0036] (f) determining the concentration of the target analyte in theexternal media from the potentiometric sensor response as compared withthe control electrochemical reference electrode.

[0037] In yet further aspect, an object of the present invention is toprovide a method for intermittently or continuously conductingcompetitive immunoassay measurements wherein a plurality of differentmeasurements are available for a single electrode without requiringregeneration of said electrode. In the situation where the amount oftarget analyte changes to become more or less concentrated in acontinuous monitoring process, a competitive binding immunoassay can beutilized. In particular, such object is accomplished by a method ofdetermination the variations in amount of a target analyte based ondisplacement activity of the enzyme-labeled detection compound (i.e.,labeled analyte) or from a binder for the analyte and the detectioncompound which binder is on the working electrode and correspondingpotentiometric responses to such attachment changes, which methodcomprises the following steps:

[0038] (a) immersing of the intermediate and/or continuousbioelectrocatalysis immunoassay sensing element (described above) in aassay medium which may vary continuously with respect to theconcentration of the target analyte present,

[0039] (b) allowing the target analyte of the assay medium to diffusethrough the diffusion membrane of the sensing device and travel to thesurface of the working electrode,

[0040] (c) permitting an immuno-equilibrium to be established within thesensing device with respect to the amount of target analyte present inthe assay medium due to (i) displacement of some or all of a labeleddetection compound (e.g., labeled analyte) that is reversibly bound to abinder immobilized on the surface of the electrode by the target analytewhich target analyte becomes reversibly bound to the binder immobilizedon the surface of the sensing element upon displacing the labeleddetection compound, or (ii) displacement of some of the reversibly boundtarget analyte from the binder on the surface of the electrode by someof the fixed amount of the labeled detection compound that is presentwithin the sensing device due to a lowering of the concentration of thetarget analyte which causes a shift in its binding equilibrium anddegree of binding with the binder on the surface of the electrode,

[0041] (d) measuring at least one shift of the electrode potentialcaused by the displacement occurring on the surface of the workingelectrode of either the reversibly bound labeled detection compound orthe reversibly bound target analyte and resulting changes inelectrocatalytic properties occurring at the surface of the electrode,

[0042] (e) determining the potentiometric sensor response which isproportional to said binding displacement on the surface of the workingelectrode,

[0043] (f) determining changes in the concentration of the targetanalyte in the external media from changes in the potentiometric sensorresponse as compared with the control electrochemical referenceelectrode and prior measurements from the working electrode.

[0044] In accordance with one aspect of the invention, there is providedan assay procedure wherein a sample containing analyte (a substance tobe determined) placed in contact with an electrode which supports abinder for the analyte, which binder has bound thereto a labeled form ofthe analyte in which the label is an electrocatalytic enzyme (an enzymethat acts on a substrate by obtaining electrons from the electrode) andwherein such electrode is also contacted with a substrate for suchenzyme label. The analyte in the sample displaces some or all of thereversibly bound labeled analyte from the binder, with the amount ofbinding displacement being directly related to the amount of analyte inthe sample. Displacement of labeled analyte results in a change involtage between such electrode and a reference electrode and such changein voltage is a measure of the amount of analyte in the sample.

[0045] In a preferred embodiment, the electrode having supported thereonthe binder having bound thereto a labeled analyte is present in achamber which includes a member or wall which is permeable to analytebut which is impermeable to the enzyme labeled analyte.

[0046] The sensing element is comprised of an electrode made byelectrochemically inert electroconductive material (such as differentcarbon based materials, gold, different electroconductive polymers)modified by immobilized binder (such as the analyte) that is directlyattached to the surface of the electrode and may be the same as theanalyte or as a detection compound.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIG. 1A illustrates an electron transfer at the surface of anelectrode. The electrode has an antigen (marked “Ag”) affixed to theelectrode as its support and a labelled antibody/enzyme complex (marked“Ab” and “Enzyme”) is attached to the affixed antigen. Due to theproximity of the enzyme to the electrode an electron is transferred tothe substrate and electrocatalytically the electrode potential ischanged by such electron transfer. Also shown in the drawing, bydiscontinuous lines and a rough raised surface is a semipermeablemembrane across which an antigen may be diffused.

[0048]FIG. 1B illustrates a background electrode potential when theantigen (marked “Ag”) which is affixed to the electrode is not linked toa labelled antibody/enzyme complex (marked “Ab” and “Enzyme”). isattached to the affixed antigen. When the enzyme is in near proximity tothe electrode an electron is transferred to the substrate andelectrocatalytically the electrode potential is changed by such electrontransfer. However, when the labeled antibody/enzyme complex becomesdisplaced (as shown in this drawing) and the enzyme is distanced fromthe electrode, no catalytic electron transfer occurs. Also shown in thedrawing, by discontinuous lines and a rough raised surface is asemipermeable membrane across which an antigen may be diffused. A doublearrow shows one such opening through which an antigen (analyte) candiffuse, but across which the antibody/enzyme complex cannot move.

[0049]FIG. 2 illustrates an immunosensor apparatus according to theinvention comprising an electrochemical working electrode 1; anelectrochemical reference electrode 2; the diffusion membrane 3 whichseparates working electrode 1 from the external media; the internalmedia 4 which may be a liquid or gel containing a substrate for anenzyme-label as shown in FIG. 1A; an external housing member 5 whichfixes together the electrochemical working electrode 1, theelectrochemical electrode 2, the diffusion membrane 3, the measuringdevice (the electrochemical interface) 6 which may be described as adigital voltmeter or an interface to microprocessor which measuringdevice is connected to the sensing element comprising theelectrochemical working electrode 1 and the supporting electrochemicalreference electrode 2, and signal recorder 7 which signal recorder maybe described as an X-T recorder, a microprocessor based data acquisitionsystem with digital display, or a personal computer.

[0050]FIG. 3 is a shematic illustrating continuous immunoassaymonitoring. In the presence of the analyte in the external media, theanalyte diffuses through the membrane and forms a complex with anincrease in potential. A decrease in the analyte concentration in theexternal media results in dissociation of the complex and leads to adecrease in the electrode potential.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0051] The present invention provides an alternative approach to thedevelopment of immunosensors and is associated with bio-electrocatalyticdetection of the reversibly displacement of a labeled detection compoundthat is reversibly bound to a binder (which binder is an analyte or acompound which will bind to the analyte) on an electrode surface, byfree target analyte which will either bind to the binder on the surfaceof the electrode or compete with the binder for binding to the detectioncompound. In this case, the electrode is separated from the externalmedia by porous membrane permeable for analyte and impermeable to highermolecular weight compounds such as the enzyme-labeled detectioncompound. Therefore, the labeled detection compound does not diffuse tothe external media. A decrease in the concentration of target analyte inexternal media results in a new (target analyte)/(labeled detectioncompound) ratio and a new equilibrium with respect to how much of theenzyme-labeled detection compound is bound to the binder on theelectrode. Such a decrease leads to release of analyte that is bound toeither the binder or to the detection compound and to the re-binding ofsome labeled detection compound to the binder on the surface of theelectrode. Therefore, continuous monitoring of the concentration of theanalyte in a sample source is allowed (FIG. 1).

[0052] As shown in FIG. 2, the working electrode 1 serves forpotentiometric measurement. It consists of an electro-conductivematerial modified by the immobilized analyte. The referenceelectrochemical electrode 2 can be fixed inside the inert body 5 or beimmersed into it together with sensing element. It can be represented byhigh impedance voltmeter or by interface which converts voltage signalto the format suitable for acquisition by microprocessor or personalcomputer. The signal recorder 7 serves to measure and visualize theinitial rate of the increase of the electrode potential of the sensingelement. It can be represented by simply X-T recorder, or bymicroprocessor based digital data acquisition system, or by personalcomputer supported with special software.

[0053] The advantage of the sensing device having a semipermeablemembrane lies in its potential to provide continuous on line monitoringof analyte. The ability of the sensing device to detect analyte over awide concentration range allows its use for on-line measurements ofconcentration for different analytes. FIG. 1 presents the overall schemeof the electrode function.

[0054] The working electrode is represented by an electro-conductivehighly dispersed material such as flat or dispersed carbon, graphite,carbon black, conductive dispersed pyrolytic products, conductive metaloxides, metal and metal powders, semiconductor materials, dispersedconductive polymers with binders (analytes or corresponding antibodies,antigens, or haptens for such analytes) that are immobilized on thesurface of the electrode material.

[0055] The liquids used in the invention are water, buffer solutions,and aqueous sample diluents. Preferred liquids are buffer solutions suchas phosphate buffered saline, borate buffered saline, acetate bufferedsaline, TRIS saline and the same buffer solutions containing detergentssuch as Tween, Triton etc. in different concentrations.

[0056] The enzyme-labels used in the invention are electrocatalyticallyactive oxidoreductases represented by but not limited to laccase(substrate oxygen); lactate dehydrogenase (substrate lactate);horseradish peroxidase, cytochrome c peroxidase, fungal peroxidases,lactoperoxidase, microperoxidase, chloroperoxidase (substrate hydrogenperoxide); hydrogenase (substrates hydrogen, proton); D-fructosedehydrogenase (substrate fructose), methylamine dehydrogenase (substratemethylamine); flavocytochrome c552 (substrate sulfide); succinatedehydrogenase (substrates succinate, fumarate); fumarate reductase(substrate fumarate); alcohol dehydrogenase (substrate ethanol);D-gluconate dehydrogenase (substrate gluconate); cellobiosedehydrogenase (substrate cellobiose); and ascorbate oxidase (substrateoxygen).

[0057] In a preferred aspect, the invention relates toimmunoelectrochemical analysis employing laccase as the enzyme label.Laccase possesses strong electrocatalytic properties in that it cancatalyze the electroreduction of oxygen (Berezin et al., Doklady Phys.Chem., 240:455 (1978, translated from Russian):

O₂+4H⁺+4e⁻→2H₂O

[0058] When laccase is utilized as the enzyme label the donor substrateis an electron moving directly from the an electrode which is in theproximity of the laccase enzyme and the second substrate is molecularoxygen.

[0059] The ability of the enzyme laccase to catalyze electroreduction ofoxygen via a direct mechanism allows the detection of the biospecificinteraction of a laccase-labeled receptor, or antibody, with aligand-modified electrode. The potential established on the electrodecoated with immobilized laccase is close to the equilibrium oxygenpotential, and the shift in potential occurring in the presence of theimmobilized enzyme can be as high as 400 mV. The bioaffinity interactionoccurring on the electrode surface can therefore be determined by usinga laccase-labeled bioconjugate. Formation of a complex between thelaccase-labeled antibody and the antigen on the electrode surfaceresults in a considerable (>300 mV) change in the electrode potential.

[0060] The change in electrode potential is due to the transfer ofelectrons directly form the electrode as a reaction substrate forlaccase which react with the other substrate for laccase which isatmospheric oxygen, in that electrons are transferred directly from theelectrode to the active site of the enzyme label. A composite carbonmaterial containing a polyethyleneimine-based polymer can be used toeliminate nonspecific interactions between the reaction components andthe electrode surface.

[0061] The potentiometric detection of an analyte does not depend on theelectrode surface area. The electrode response is a function of thefraction of electrode surface that is covered by the electro-activelabel (laccase). This fact opens a possibility for miniaturizationimmuno-electrodes without effecting of their sensitivity. One of theimportant advantages of the bio-electrocatalytic detection approach inimmunoassay is associated with the fact that the reaction does notinvolve low molecular weight substrates (except dissolved oxygen).

[0062] Several general practical advantages of the potentiometricimmuno-sensors according to the invention can be summarized in fiveengineering issues: (i) potentiometric detection has a potential for agreat degree of miniaturization of sensing elements; (ii) themanufacturing procedure of the sensing elements can easily be adaptedfor mass production being compatible with techniques such asscreen-printing or ink-jet dispensing; (iii) it is possible to designlow cost and thus, disposable sensing elements; (iv) the measuringequipment is simple consisting of a high impedance voltmeter.

[0063] In a preferred aspect, the invention provides a potentiometricimmunosensor based on mediatorless bioelectrocatalysis that utilizes thelaccase enzyme as an electrocatalyst-label. The electrocatalyticproperty of the enzyme in the reaction of oxygen electroreduction(reaction 1) allows the detection of the bio-specific interaction of alaccase-labeled receptor, or antibody, with a ligand modified electrode.Analysis was performed in a competitive scheme, and a single measurementwas made within 20 minutes. Such a potentiometric immunoassay does notrequire an electrochemically active mediator. The reaction substratesare atmospheric oxygen and electrons that are transferred directly fromthe electrode to the oxygen molecule via the active site of the enzyme.Insulin may be used as a model analyte as illustrated by the examplesbelow. Furthermore, a higher rate of electrode potential shift can beachieved by employing a high concentration of immuno-conjugate. Acompetition for binding with immobilized immuno-species for highconcentration of immuno-conjugate can require a relatively highconcentration of the analyte.

[0064] In one aspect of the invention the laccase enzyme label is boundto a binder (antibody, antigen or hapten, that is referred to above as adetection compound) for an analyte which detection compound is alsobound by a binder on the electrode. The analyte will cause the enzymelabel/detection compound to disassociate from the binder on theelectrode and bind to the analyte to cause a change in potential of theelectrode. Depending upon dynamic changes in the concentration ofanalyte that moves across the semipermeable membrane of the sensor whichcontains the enzyme label/ligand and electrode, differing measurementsof electrode potential are observed. For example, at higherconcentrations of analyte more of the enzyme label/detection compound isdisassociated from the electrode to bind to the analyte and at lowerconcentrations of the analyte more of the enzyme label/detectioncompound binds to the binder on the electrode. Since measurements of theincrease or decrease in electrode potential can be made simultaneouslywith the biospecific interaction of the affinity components on theelectrode surface, continuous measurements can be performed in thekinetic mode. This also allows for a significant reduction in analysistime since the rate of change in potential can be utilized to determinethe changes in concentration as well as equilibrium measurements ofpotential.

[0065] In general, the amplitude of signals produced by workingelectrodes according to the invention is determined by the extent towhich the electrode surface is coated with laccase-conjugated molecules,and not by the total amount of conjugated laccase bound to theelectrode. A carbon composite can be used as an electrode material whichpossess a considerably large effective surface area, but highersensitivity can be attained by the use of an electrode materials with alower, or low, effective surface. The pH of the assay solution must behigh enough to promote antigen-antibody complex formation. At the sametime, it should not exceed the optimal value for the enzymatic activity,which is the case of laccase activity is about pH 5.5, but may exceedthe optimal value for laccase activity somewhat. For example, anincrease in pH above 5.5 when using laccase can result in a sharpdecrease in the rate of potential change in the presence of thelaccase-antibody conjugate, but may be as high as 6.5 where laccase fromCoriolus sp. still retains 30-50% of maximal activity. Since laccasefrom different sources may differ in pH for their optimium activity, alaccase may be selected from a particular source when a particularoptimal pH is desired. For example, laccase from certain fungi has a pHoptimal activity near 7.0. Electrodes according to the invention, asdescribed above do not have changes in the increase of the electrodepotential over the temperature range of 30° C.-37° C., and dryIgG-modified electrodes will generally retain their activity duringstorage for at least 2 months at 4° C.

[0066] In another aspect the invention provides a method and apparatusfor reagentless immunoassay is described, in that no reagent beyond theself-contained sensor apparatus is required and the self-containedsensor apparatus may be used for multiple measurements.

[0067] In a preferred embodiment of the invention the apparatuscomprises an immunosensor have the general immunosensor design shown inFIG. 2, which includes the working electrode 1; the referenceelectrochemical electrode 2; a diffusion membrane which separates theworking electrode 1 from the external media; the internal media 4 whichis liquid or gel containing a substrate for an enzyme-label; an externalhousing member 5 which fixes together the working electrode 1, theelectrochemical reference electrode 2, the diffusion membrane 3, themeasuring device (the electrochemical interface) 6 which is a digitalvoltmeter or an interface to microprocessor which is connected to thesensing element and the supporting electrochemical probe 2, and signalrecorder 7 which is an X-T recorder, microprocessor based dataacquisition system with digital display, or personal computer. Theworking electrode 1 serves for potentiometric measurement. It consistsof an electro-conductive material modified by the immobilized analyte.The supporting electrochemical reference electrode 2 can be fixed insidethe external housing member 5 or be immersed into it together with thesensing element. It can be represented by high impedance voltmeter or byinterface which converts voltage signal to the format suitable foracquisition by microprocessor or personal computer. The signal recorder7 serves to measure and visualize the initial rate of the increase ofthe electrode potential of the sensing element. It can be represented bysimply X-T recorder, or by microprocessor based digital data acquisitionsystem, or by personal computer that is set to record and analyze themeasurement results.

[0068] The above described apparatus may have multiple electrodes wherethe different electrodes are for different analytes. Thus, multipleanalytes in a single sample may be analyzed for by utilizing themultiple electrodes.

[0069] Further, a Sandwich Assay Scheme can be used for continuousimmunoassy monitoring. In the case where both primary and secondary(labeled) antibody are low affinity antibodies, the formation of acomplex Ab1-Analtye-Ab2-Laccase may be reversable.

[0070] Having described the invention, the following non-limitingexamples are given to illustrate specific techniques and applications ofthe principles of the invention. which can be used to carry out theinvention. These specific examples are for illustrative purposes onlyand are not intended to limit the scope of the invention described inthis application.

EXAMPLE 1 Preparation of a Laccase Enzyme Conjugate

[0071] Laccase from Coriolus sp. was obtained as described by Ghindiliset al., Biochemistry (translated from Russian: Biokhimiya) 53:635-639(1988). Pig insulin was obtained as a commercial product (for example,Sigma, St. Louis, Mo., U.S.A.). The laccase-insulin conjugate wassynthesized by using the general procedure of Nakane et al., J. Hist andCyto. Chem., 22 1084-1091 (1974) for the preparation of peroxidaseconjugates. This procedure was modified as follows: a solution of NaIOwas added to the solution of laccase in distilled water (1 mg ml⁻¹) togive a final concentration of 1.0 M. The mixture was incubated for 30minutes, in the dark, at 18-22° C., and dialyzed against 0.1M sodiumacetate buffer, pH 4.5 at 4° C. for 14-16 hours. Insulin was graduallyadded to the enzyme solution to give a molar ratio of 3:2. The mixturewas incubated for 3 hours at 18-22° C. The pH of the reaction medium wasmaintained between 8.8 and 9.0. The conjugate obtained was dialyzedagainst 0.1 M phosphate buffer, pH 6.5, at 4° C., for 16-18 hours, andstored in 50% glycerol at −18° C. EXAMPLE 2

Preparation of a Working Electrode

[0072] Graphite Ink, EXP 741801, obtained by Ercon (Wareham, Mass.), wasdeposited on a plastic strip (2×30 mm) and then dried at roomtemperature for 14-16 h. The electrode body was then encapsulated withfast dry enamel 720, obtained from Maybeline Inc. (New York, N.Y.). Thetips of the electrode (2×2 mm) remained non-encapsulated to serve as aworking electrode surface and as a connector to the electrical circuit.The electrode was then pretreated, by forced polarization, in a threeelectrode electrochemical cell under an electrode potential of −0.8 Vversus an Ag/AgCI reference electrode for 20 min. A solution ofmonoclonal anti-insulin antibodies (20 μg/ml) in phosphate bufferedsaline (PBS) was then placed onto the working tip of the electrode anddried to achieve immobilization by physical absorption. Monoclonalantibodies were developed by Biocon Inc. (Rockville, Md.), from the ATCCHB127 hybridoma line obtained from ATTC (Rockville, Md.). The electrodewas then incubated in a solution of trypsin inhibitor (0.1 mg/ml) in PBSto block free sites of non-specific binding for 4h at room temperature.The electrode was then pre-incubated in the solution of laccase-insulinconjugate (0.1 mg/ml) in 0.1 M phosphate buffer, pH 6.2, containing 1mg/ml human serum albumin (HSA) at 37 C., for 16 h, to achieve anattachment of the laccase-insulin conjugate to the electrode surface.

EXAMPLE 3 Analysis of Analyte Insulin Solution With a Working Electrode

[0073] The electrode was then placed into the sensing apparatus, asdescribed in FIG. 2. The internal medium of the housing member containeda solution of laccase-insulin conjugate (5 μg/ml) in 0.1 M phosphatebuffer, pH 6.2, containing 1 mg/ml HSA. The sensing apparatus was thenplaced in a contact with an external media (0.1 M phosphate buffer, pH6.2, containing 1 mg/ml HAS) containing insulin analyte.

[0074] Atmospheric oxygen, which is a substrate for laccase, diffuses tothe internal media through the external media. The potential of theworking electrode, in the absence of insulin in the external media(antibody/laccase complex is bound to insulin that is affixed to theelectrode) was close to O₂/H₂O potential and was about 350 mV (vsAg/AgCl electrode). The addition of insulin into the external mediaresulted in a shift of the potential towards the background carbonelectrode potential (100 mV). The potential change was proportional tothe concentration of insulin in the external media in a wide range ofinsulin concentrations.

[0075] As a reference, an Ag/AgCl electrode was used. Potential changeswere measured by means of a high impedance voltmeter.

What is claimed is:
 1. A method for continuous determination of a targetanalyte in a sample comprising the steps of: (a) contacting an analytewith an immunosensory apparatus comprising a working electrode having abinder on its surface and a reference electrode and wherein said workingelectrode requires no regeneration between consecutive measurements ofthe analyte; (b) determining a change in the working electrodepotential, and (c) comparing the potentiometric response of the workingelectrode with the reference electrode wherein a difference indicatesthe presence of analyte in said sample and said difference isproportional to the concentration of analyte, and wherein saidmeasurement can be performed with a single working electrode, therebydetermining the concentration of said analyte in the sample.
 2. Themethod of claim 1 wherein said analyte is selected from the groupconsisting of an antigen, a hapten and an antibody.
 3. The method ofclaim 1 wherein said labeled detection compound is selected from thegroup consisting of an antigen, a hapten and an antibody.
 4. The methodof claim 1 wherein said labeled detection compound is the same as theanalyte.
 5. The method of claim 1 wherein said labeled detectioncompound is a binder for the analyte.
 6. The method of claim 1 whereinthe binder is a binder for both the analyte and the labeled detectioncompound.
 7. The method of claim 1 wherein the analyte binds reversiblyto the binder.
 8. The method of claim 1 wherein said electrocatalyticenzyme an oxidoreductase.
 9. The method of claim 1 wherein saidelectrocatalytic enzyme is a member selected from the group consistingof laccase, lactate dehydrogenase, horseradish peroxidase, cytochrome cperoxidase, a fungal peroxidase, lactoperoxidase, microperoxidase,chloroperoxidase, hydrogenase, D-fructose dehydrogenase, methylaminedehydrogenase, flavocytochrome c552, succinate dehydrogenase, fumaratereductase, alcohol dehydrogenase, D-gluconate dehydrogenase, cellobiosedehydrogenase and ascorbate oxidase.
 10. The method of claim 1 whereinsaid electrocatalytic enzyme is laccase.
 11. The method of claim 1wherein the diffusion medium is a liquid or a gel.
 12. The method ofclaim 1 wherein said diffusion medium contains a second substrate forthe electrocatalytic enzyme and the analyte binds to one of either thebinder on said working electrode or the detection compound.
 13. Themethod of claim 12, wherein said second substrate is oxygen.
 14. Amethod for determining a target analyte in a sample comprising the stepsof: (a) contacting an analyte with an immunosensory apparatus,comprising a diffusion membrane, a reference electrode and a workingelectrode, the latter having on its surface a binder with a labeleddetection compound attached thereto, wherein said electrodes and saidmembrane are separated by a chamber containing a diffusion medium thatcontains a substrate of an electrocatalytic enzyme and wherein saidelectrodes are separated from the source of said analyte by saidmembrane, under conditions promoting diffusion of said analyte throughsaid membrane to contact said electrodes; (b) allowing the analyte todisplace the labeled detection compound from said binder whereupon saidanalyte becomes bound to the binder on the surface of the workingelectrode in the case where the detection compound is the same as theanalyte or the analyte becomes bound to the detection compound in thecase where the labeled detection compound is a binder for the analyte,and wherein said detection compound is bound to an electrocatalyticenzyme such that in the presence of the target analyte the workingelectrode provides an electron to said electrocatalytic enzyme whichprovides an electron to a substrate of said enzyme (c) determining achange in the working electrode potential, and (d) comparing thepotentiometric response of the working electrode with the referenceelectrode wherein a difference indicates the presence of analyte in saidsample and is proportional to the concentration of analyte, therebydetermining the concentration of analyte in the sample.
 15. The methodof claim 14 wherein electrode regeneration is not required betweensuccessive determinations of said analyte.
 16. The method of claim 14wherein regeneration of the working electrode and other reagents is notrequired between successive determinations of said analyte.
 17. A methodfor intermittently or continuously conducting immunoassay measurementswherein a plurality of different measurements for an analyte areavailable for a single electrode without requiring regeneration of saidelectrode and other reagents, wherein said method is for determining atarget analyte based on displacement activity of the target analyte anda potentiometric mode, said method comprising the following steps: (a)immersing of the intermediate and/or continuous bioelectrocatalysisimmunoassay sensing element of claim 8 in a assay medium containing thetarget analyte, (b) allowing the target analyte of the assay medium todiffuse through the diffusion membrane of the sensing device and travelto the surface of the working electrode, (c) permitting animmuno-equilibrium to be established within the sensing device withrespect to the amount of target analyte present in the assay medium dueto displacement of some or all of a labelled detection compound from thebinder on the surface of the electrode by the target analyte whichtarget analyte becomes bound to the binder on the surface of the sensingelement in the case where the detection compound is the same as theanalyte or the target analyte becomes bound to the detection compound inthe case where the detection compound is a binder for the analyte, (d)measuring at least one shift of the electrode potential caused by thedisplacement of some or all of the labeled detection compound from theelectrode's surface and the resulting diminishment or absence ofelectrocatalytic properties caused by the label of detection compound,(e) determining the potentiometric sensor response which is proportionalto the degree of displacement of the labeled detection compound from thebinder on the surface of the working electrode caused by competitivebinding of the target analyte, and (f) determining the concentration ofthe target analyte in the external media from the potentiometric sensorresponse as compared with the control electrochemical referenceelectrode.